The present invention is directed to methods and compositions for treating diseases mediated by transglutaminase activity, by inhibiting the activity of transglutaminase.
Protein cross-linking resulting in the formation of aggregates is a common feature of a number of neurodegenerative diseases, including Alzheimer""s disease and the family of diseases exemplified by Huntington""s Disease, caused by expansion of CAG trinucleotides encoding polyglutamine (Green, 1993; Prosiner et al, 1983; Davies et al, 1997; Scherzinger et al, 1997; DiFiglia et al, 1997).
Several neurodegenerative diseases, including Huntington""s Disease, spinobulbar atrophy (Kennedy""s disease), various spinocerebellar ataxias (SCA 1, 2, 3, 6, 7), and dentatorubralpallidoluysian atrophy (DRPLA), involve proteins with long stretches of polyglutamines in their N-terminus (Ross, 1995). Cross-linking of these polyglutamine containing proteins may be critical in the neurologic dysfunction and pathology characteristic of these disorders (Ross, 1995). Recently, nuclear inclusions containing ubiquinated aggregates of huntingtin (htt), DRPLA protein, ataxin 1 and ataxin 3, respectively, have been observed in the affected brain areas of patients with Huntington""s Disease, DRPLA, SCA-1 and SCA-3 (DiFiglia et al, 1997; Igarashi et al, 1998; Skinner et al, 1997; Paulson et al, 1997). Interestingly, both htt and ataxin 3 are primarily cytoplasmic proteins in healthy individuals.
In Huntington""s Disease, as well as in spinobulbar atrophy, various spinocerebellar ataxias (SCA 1, 2, 3, 6, 7) and dentatorubralpallidoluysian atrophy, the gene encoding the mutant protein contains expanding trinucleotide repeats of the codon CAG. These repeats encode glutamine (Q). With each ensuing generation, because of the expansion of these repeats, disease onset is earlier, a phenomenon known as genetic anticipation. There is no genetic anticipation, however, when the disease is transmitted through the female line in Huntington""s Disease. The importance of the polyglutamine domain is further emphasized by the observation that CAG repeats, ectopically introduced into an unrelated gene encoding hypoxanthine phosphoribosyltransferase (hrpt), produce a phenotype similar to that seen in the human neurologic disorders related to abnormal polyglutamine domains (Ordway et al, 1997). The length of the polyglutamine domain is absolutely critical for the appearance of Huntington""s Disease, as well as the other neurologic diseases involving mutations in genes involving expansion of CAG repeats. In Huntington""s disease, for example, if the polyglutamine domain exceeds 36 Q repeats, the fatal neurologic disease ensues. In other CAG trinucleotide repeat diseases, there is a pathologic threshold, although the length varies from disease to disease, with the shortest threshold (21Q) in SCA-6, and longer thresholds in SCA-3 (61Q) and dentatorubralpallidoluysian atrophy (49Q) (Lunkes et al, 1997).
Huntingtin is expressed at similar levels in patients with Huntington""s Disease and controls, regardless of the number of glutamine repeats. Huntingtin is also expressed throughout all tissues of the body and is expressed in equal amounts in all regions of the normal brain. In affected areas of Huntington""s disease brain, mutant huntingtin is much less abundant than wild-type huntingtin (Schilling et al, 1995; Trottier et al, 1995; Strong et al, 1993). Although the Huntington""s Disease gene is widely expressed (Huntington""s Disease Collaborative Research Group, 1993; Sharp et al, 1995), the pathology of Huntington""s Disease is restricted to the brain, and to specific regions within the brain, for reasons that remain poorly understood. At death the brain is small and often weighs less than one kilogram, as compared to the brain of a normal young adult, which weighs 1.4 kg. The frontal and parietal lobes are smaller than normal, but the most distinctive damage is visible in the head of the caudate nucleus, which is shrunken, along with the putamen and globus pallidus. The pathologic signature of Huntington""s Disease is the loss of virtually all medium spiny neurons in the caudate. The brainstem and cerebellum are normal. Microscopically, there is extensive loss of neurons in the caudate and putamen, with evidence for apoptosis and necrosis (Portera-Caillau et al, 1995).
Huntingtin is located in neurons throughout the brain, with the highest levels evident in larger neurons. Huntingtin is a cytosolic protein primarily found in somatodendritic regions (Sharp et al. 1995; Strong et al, 1993). Recently, immunocytochemrical studies, using antibodies generated against peptides corresponding to the huntingtin N-terminus, suggest that inclusions containing huntingtin are present in the nucleus of striatal neurons of Huntington""s Disease patients, but not in their cerebellar or brainstem neurons (DeFiglia et al, 1997). In the adult form of Huntington""s Disease, axonal inclusions in dystrophic neurites are far more common than nuclear inclusions (DiFiglia et al, 1997). These inclusions are never found in normal individuals. These inclusions contain aggregates of huntingtin. These inclusions do not have the appearance of amyloid: xe2x80x9csearches for amyloid deposits in brains of Huntington""s Disease patients have been negative.xe2x80x9d (Lunkes et al, 1997).
These aggregates stain with antibodies directed to the N-terminus of huntingtin but not to the C-terminus. The huntingtin N-terminal fragment, containing the polyglutamine domain, is most likely bound to ubiquitin via a lysine ubiquitin bond (Ciechanover, 1994). Somehow, in the pathogenesis of Huntington""s Disease, the mutant huntingtin translocates to the nucleus and forms inclusions composed of aggregated N-terminal fragments of huntingtin. This is a pathological feature of the disease (Davies et al, 1997; Scherzinger et al, 1997; DiFiglia et al, 1997). Recently ubiquitinated intranuclear inclusions containing expanded polyglutamine domains were also seen in neurons in dentatorubralpallidoluysian atrophy (Igarashi et al, 1998), spinocerebellar ataxia type 3 (Paulson et al, 1997) and in spinocerebellar ataxia type 1 (Skinner et al, 1997).
Two mechanisms have been postulated to explain the cross-linking of huntingtin; these mechanisms may not be mutually exclusive. Molecular modeling had shown that xcex2-strands made of polyL-glutamine can be assembled into sheets or barrels by hydrogen bonds between their main-chain and side-chain amides (Perutz et al, 1994). Perutz and colleagues (Stott et al, 1995; Perutz, 1996) tested this model experimentally. They showed that synthetic polyL-glutamine (Asp2-Q15-Lys2) (SEQ ID NO:1) forms xcex2-strands, which are held together by hydrogen bonds between their amide groups. These aggregates maintain their secondary structure at pH 7 and pH 3. Interestingly, at pH 7 the peptide gradually precipitated. They postulated that these polymers comprised of polar zippers may be responsible for the neurodegeneration seen in Huntington""s Disease. Recently, Scherzinger and colleagues showed that a glutathione S-transferase (GST) fusion protein encoding part of exon 1 of huntingtin, containing a polyglutamine domain of 51Q, spontaneously aggregates into amyloid-like fibrils, after enzymatic cleavage of the GST protein together with a few amino acids of exon 1 of huntingtin (Scherzinger et al, 1997) The GST-huntingtin Q51 construct was soluble; aggregates were formed only upon total enzymatic cleavage of the GST tag from GST-httQ51. Somehow, covalent fusion of the peptide with the polyglutamine domain to an unrelated protein, GST, prevented aggregation.
The GST-htt intermediate may serve as a nucleation factor for ordered protein aggregation in this system (Scherzinger et al, 1997). Indeed, this model is supported by the experimental finding of intermediate structures, termed xe2x80x9cclotsxe2x80x9d, on one or both ends of the growing fibrils. Scherzinger and colleagues stated, xe2x80x9cThese clots were not detected when GST-httQ51 was digested to completion with trypsin, which totally degrades the GST tag, while they were detectable upon limited digestion, leaving the GST moiety intact. This indicates that these structures are transient intermediates.xe2x80x9d Expression of a GST-htt fusion protein may, thus, have allowed the GST to act as an intermediate, allowing for the aggregation of htt.
Green (1993) proposed a second hypothesis to explain huntingtin aggregation. Green suggested that polyglutamine tracts above a certain pathologic length become better substrates for transglutaminase. The resulting aggregated huntingtin, either cross-linked within itself or with other proteins, then becomes toxic for neurons.
Transglutaminases are a family of ca2+ dependent enzymes that catalyze the formation of isopeptide bonds between the side chains of glutamine and lysine (K) residues. When a protein-bound K residue serves as the primary amine donor, the reaction results in the formation of an xcex5-(xcex3-glutamyl)-Lys isopeptide bond that serves to cross-link the proteins (Green et al, 1993; Folk, 1980). In addition to proteins containing lysine, the polyamines spermidine and spermine may serve as substrates for transglutaminases. The resulting bond is covalent, stable and relatively resistant to proteolysis (Folk, 1980). This cross-linking occurs between two glutamine residues (in the presence of a diamine) or between one glutamine residue and a K residue. When such a bridge is formed between two glutamine residues, it is possible that an adaptor molecule provides the diamine donor which is involved. It is postulated that transglutaminases promote cross-linking between various domains within the Huntington""s Disease protein and other cellular proteins.
Little is known about how huntingtin interacts with itself or with other proteins. Kahlem et al showed that polyglutamine peptides, when flanked by adjacent amino acids from the residues found in the proteins associated with SCA-1, SCA-3, and dentatorubralpallidoluysian atrophy (DRPLA), or flanked by arginine, could serve as a substrate for arginine transglutaminase (RTGase) (Kahlem et al, 1996). Peptides with Q greater than 18 could not be used in those studies, because of their instability and their tendency to form spontaneous aggregates. In the presence of a brain extract from rat containing transglutaminase activity and R5Q18R5 (SEQ ID.NO:2), as glutamine acceptor, and a rat brain protein fraction as amine donor, brain proteins were aggregated due to the endogenous transglutaminase activity in the extract (Kahlem et al, 1996).
Although they did not work with GST constructs of huntingtin, Cooper et al showed that either GST-Q10 or GST-Q62 could serve as substrates for tissue transglutaminase (Cooper et al; 1997). Previously, they had shown that huntingtin and the DRPLA protein bind selectively to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in brain homogenates (Burke et al, 1996). GAPDH in brain homogenates bound to an immobilized Q60 polypeptide, but not to an immobilized Q20 peptide. Moreover, transglutaminase could inhibit GAPDH to a greater extent in the presence of GST-Q62 or GST-Q81 than in the presence of GST-10. These experiments imply that polyglutamine domains disrupt cerebral energy metabolism after aggregation with transglutaminase.
Insulin-dependent diabetes mellitus (IDDM) in NOD mice and mouse experimental autoimmune encephalomyelitis (EAE) are the major disease models for human type I diabetes and multiple sclerosis, respectively. Compared with the Interleukin-2 (IL-2) protein produced by B6 mice, NOD-produced IL-2 shows differences in glycosylation that may affect its functional half-life. If the NOD/SJL allele of IL-2 influences EAE and diabetes susceptibility, a possible mechanism may lie in its role in T-cell selection in the thymus or in its function in the peripheral immune compartment. Insufficient levels of IL-2 may affect negative selection in the thymus, allowing the escape of self-reactive T-cells. IL-2 is also important in the autocrine feedback loop that regulates the expansion of antigen-specific T-cell clones by inducing apoptotic cell death, and is essential for the maintenance of self-tolerance as evidenced by the development of severe autoimmunity in IL-2 mice (Encinas et al., 1999).
It is an object of the present invention to overcome the aforesaid deficiencies in the prior art.
It is another object of the present invention to inhibit in vivo the activity of transglutaminase.
It is a further object of the present invention to treat neurological diseases involving aggregation of polyQ proteins, such as huntingtin.
It is another object of the present invention to treat neurological diseases presenting aggregated polyQ proteins by inhibiting the activity of transglutaminase.
It is a further object of the present invention to treat diseases mediated at least in part by transglutaminase by administering an inhibitor for transglutaminase.
It is another object of the present invention to treat cell-mediated autoimmune diseases by administering an inhibitor of transglutaminase.
It is a further object of the present invention to treat diseases characterized by inflammatory infiltrates in the central nervous system by inhibiting the activity of transglutaminase.
It is another object of the present invention to treat multiple sclerosis by inhibiting the activity of transglutaminase.
Neurodegenerative diseases involving cross-linking of polyQ proteins, resulting in the formation of aggregates, can be treated by inhibiting the action of transglutaminase. Treatment includes reversing ongoing paralysis as well as lymphocytic infiltration in the brain. This inhibition can be effected by administering to a patient in need thereof an effective amount of a compound which inhibits the activity of transglutaminase, thereby inhibiting or reversing cross-linking of the polyQ proteins. Compounds which have been found to inhibit transglutaminase activity include monodansyl cadaverine, monoamines and diamines such as cystamine, putrescine, GABA (gamma-amino benzoic acid), N-benzyloxy carbonyl, 5-deazo-4-oxonorvaline p-nitrophenylester, glycine methyl ester, CuSO4, and the oral anti-hyperglycemic agent tolbutamide.
The activity of transglutaminase can also be inhibited by means of gene therapy. By this means, a DNA sequence which inhibits or prevents the activity of transglutaminase, or which encodes a polypeptide which inhibits or prevents the activity of transglutaminase, can be delivered directly to the cells of interest. Such a substance may be a DNA or RNA sequence which is antisense to the transglutaminase gene, thereby preventing its transcription and expression. Alternatively, the DNA delivered to the cells of interest may encode a polypeptide which is an inhibitor of transglutaminase or which otherwise prevents the activity of transglutaminase. Such a polypeptide may be an antibody, including a single chain antibody or the antigen binding domain of an antibody, which will bind to transglutaminase and thereby inhibit its activity. A short peptide which is a substrate for transglutaminase and therefore prevents its action on the polyQ protein may also be used. Such a peptide can readily be designed by one of ordinary skill in the art.
Additionally, because interleukin-2 is a polyQ molecule, cell-mediated autoimmune diseases can be created by inhibiting transglutaminase activity by any of the methods disclosed herein and thus inhibiting crosslinking of interleukin-2. Such diseases include multiple sclerosis, rheumatoid arthritis, and insulin dependent diabetes mellitus.
Because transglutaminase is critical for adherence of activated lymphocytes to inflamed brain endothelium and for the subsequent passage of lymphocytes into the central nervous system, inflammatory diseases of the central nervous system can be treated by inhibiting transglutaminase activity by any of the means disclosed herein.